Human (-defensins are a family of 3-4 kDa, cationic and Cys-rich antimicrobial proteins expressed predominantly in leukocytes and epithelial cells. They kill a broad range of microbes presumably through disruption of the negatively charged microbial membrane, playing critical roles in phagocytosis and in mucosal protection against invading pathogens. While an important component of innate immunity, human (-defensins also function as effective immune modulators in adaptive immunity by chemoattracting monocytes, subsets of T lymphocytes and immature dendritic cells. Recently, three human neutrophil (-defensins (HNP1-3), differing from each other by a single amino acid residue at the N-terminus have been shown to suppress HFV-1 infection in vitro. We have found that the distantly related fourth member of human neutrophil a-defensins (HNP4) and two human intestinal alpha-defensins (HD5 and HD6) are also effective inhibitors of HIV-1 replication in peripheral blood mononuclear cells. Despite the prominent roles alpha-defensins play in both innate and adaptive immunity and their potential therapeutic value in the treatment of infectious diseases, the structure/function relationships for these antimicrobial proteins have not yet been explored. The sequence rules and structural determinants that govern the great variety of biological functions of alpha-defensins and mechanisms of their action continue to remain poorly understood. Such study has been hindered, in part, by the difficulty producing recombinant defensins due to their inherent antibiotic and membranolytic properties. On the other hand, chemical synthesis of alpha-defensins, in spite of their small size, is technically challenging due to known problems associated with oxidative folding. Consequently, a significant body of the published work on alpha-defensins has been based largely on studies of HNP1-3 purified from neutrophils. As the other three native alpha-defensins have been recovered in amounts that are small (HNP4), smaller (HD5), or nil (HD6), considerably less is known about their properties. Our laboratory has recently developed a robust synthetic approach to, and an efficient folding protocol for, the production of all six human alpha-defensins in high purity and yield, enabling for the first time a systematic and comparative structure/function relationship study on these antimicrobial proteins. Such study will not only provide a better understanding of how defensins function at the molecular level, but more importantly, yield valuable insights into designing defensin-based novel therapeutic agents to combat infectious microbes. Specific aims envisioned in this application are as follows. Specific Aim 1: Elucidate the mechanism of inhibition of HNPs by their pro peptides. Specific Aim 2: Dissect the molecular determinants for the antimicrobial activity and specificity of alpha-defensins. Specific Aim 3: Decipher the physiological relevance and functional importance of HNP oligomerization.